Direct cell-to-cell diffusion of ions and cytosolic molecules is mediated by gap junction (GJ) channels. Each channel is a multimer of connexin (Cx) subunits that forms an intercellular aqueous pore b docking two pre-assembled hemichannels, one from each of two apposed cells. The first aim of this proposal is to study GJ channel formation as it relates to the events that occur after the establishment of cell-cell contact We utilize our ability to manipulate cells into contact while recording and uniquely combine electrophysiology of GJ formation with fluorescence imaging of Cx distribution in vivo real time. Cxs will be fused with enhanced GFP to allow their visualization. We will examine how the dynamics of Cx redistribution, such as plaque formation is associated with the establishment of electrical coupling. We will examine whether Cxs arrive at the junctional membrane from cytoplasmic stores of adjacent membranes and how membrane fluidity and extracellular Ca2+ affect the association between Cx distribution and GJ formation. We will also study the biophysical properties of de novo GJ channel openings, a process that we propose is a form of gating elicited by hemichannel docking. The second aim of the proposal is to test the hypothesis that the de novo opening of a GJ channel involves Cx domain(s) that are part of a ~common~ gate that is acted upon by different agents. Based on our studies of voltage and chemical gating of GJ channels formed of different Cxs, activators of this common gate include alkanols, H+ and membrane voltage. These agents close GJ channels completely, and the ~common gate~ may represent the principal means by which coupling is dynamically regulated in cell populations where voltages remain uniform. We will also examine gating by transjunctional voltage (Vi). We demonstrate that the Vi gate only partially closes GJ channels to a residual state whose conductance is Cx specific. We will examine the ionic selectivity of the residual state and will assess the pore size of the residual and fully open states by measuring intercellular diffusion of uncharged dyes. The third aim of this proposal is to explore how Vi gating combined with the rectifying properties of open and residual states can play a role in regulating electronic transmission between excitable cells. Using heterotypic junctions, with combinations of Cxs that are present in the nervous system, we demonstrate conditions under which some heterotypic junctions exhibit nearly unidirectional transmission. These studies focus on defining the mechanisms that control GJ channel formation and gating which are ultimately necessary for determining the role of intercellular communication in normal and diseased states. Human genetic disorders that have been linked to Cx dysfunction include C-linked CMT disease, visceroatrial heterotaxia and sensorineural deafness.